Planet Biotechnology Inc. (PBI) has pioneered methods for the production of functional therapeutic antibodies and immunoadhesins in transgenic tobacco. For reasons that are unclear, the yield of antibodies in transgenic plants has generally been low, with most published literature citing maximum expression of IgG in transgenic tobacco well below 30 mg/kg. We have designed and investigated the expression in plants of human IgG1 variants using DNA sequences in which there are over- or under-represented codon pairs. By utilizing an IgG heavy chain constant region containing many over-represented codon pairs we were able to significantly enhance transient expression of a number of antibodies (up to 35-fold higher than with our codon usage optimized sequence). Furthermore, we have generated stable tobacco plants expressing up to 515 mg IgG/kg fresh weight. Evidence suggests that the more over-represented a codon pair is, the more slowly it is translated. Local translational pausing might provide a time delay, enabling sequential folding of defined portions of nascent polypeptides, which may prevent protein misfolding and aggregation, and reduce degradation. We will test the hypothesis that translation is attenuated at over-represented codon pairs, and that translational pauses at particular positions are necessary for efficient co-translational folding and assembly of immunoglobulins in plants. To determine whether the expression benefit of the presence of many over-represented codon pairs is particular to IgG heavy chain in plants, or whether it is more widely applicable, sequences will be designed and synthesized for a small protein (J chain) and a larger multi-domain protein (IgA2 heavy chain). Furthermore, the IgG1 heavy chain, IgA2 heavy chain and J Chain sequences will be designed for expression in tobacco, in which the positions of over-represented codon pairs in the expression host are harmonized with those in the native host. The expression-competence of these sequences will be established using a tobacco transient expression system, after which stable transgenic tobacco plants will be generated. Specific transcript accumulation levels will be measured by real-time PCR and compared to protein accumulation levels. Endogenous BiP mRNA levels (a molecular marker of the unfolded protein response) will be quantified by real- time PCR in tobacco plants expressing the genes described above. 35S-pulse-chase labeling assays will determine the influence of over- or under-represented codon pairs on protein translation and turnover rates of the genes described within the grant. If successful, the result will be a general platform technology for the quick, easy, scalable production of therapeutic human antibodies in large quantities from plants (and possibly other eukaryotic expression hosts). PUBLIC HEALTH RELEVANCE: Planet is developing tobacco-produced recombinant proteins against several diseases including: intoxication by Botulinum Neurotoxin A (BoNT/A), the most potent toxin known and a bio-terror agent, the common cold, dental caries and intoxication by Anthrax toxin, also a bio-terror agent. All of these product-candidates could benefit from a technology that would increase expression in plants, which would result in decreases in production and purification costs, making therapeutic antibodies more affordable and widely available.